In about 75 percent of metastatic breast cancer patients, bone is the preferred site for metastasis. The Hh signaling pathway is aberrantly activated in breast cancer. Our laboratory has investigated the role of the Hedgehog (Hh) pathway as a determinant of metastasis of breast cancer to the bone. We have observed a novel crosstalk between breast cancer cells, osteoblasts and osteoclasts via the Hh pathway that results in bone resorption. We further noted that inhibition of Hh signaling in breast cancer cells resulted in decreased tumor biomass in the femur and tibia of athymic mice injected with breast cancer cells. Bone is a hypoxic microenvironment (pO2 between 1-7%). Under hypoxia, cells switch from aerobic to anaerobic metabolism to meet the energy requirements for survival. Thus, tumor hypoxia selects for cells dependent on anaerobic metabolism. This increases the tumor biomass comprising of metastatic tumor cells adapted for survival under hypoxic conditions (Note: Anaerobic metabolism is distinct from the Warburg effect, which is aerobic glycolysis). Our objective is (i) to evaluate the mechanism(s) of hypoxia-induced resistance of breast cancer cells to Hh inhibitors and, (ii) to establish the therapeutic benefit of impeding the hypoxia response of breast cancer cells to enhance their sensitivity to Hh inhibitors We hypothesize that the hypoxic environment in the bone and the consequent hypoxic response of breast cancer cells activates non-classical Hh signaling in the tumor cells making them resistant to Hh inhibitors. Our hypothesis will be tested in studies described in the following SPECIFIC AIMS: Specific Aim 1: Elucidate the mechanism(s) by which Hh signaling impacts the ability of breast cancer cells to adapt to hypoxia (anaerobic metabolism) and induce osteolysis. Specific Aim 2: Define the mechanism(s) of hypoxia-induced non-classical activation of Hh signaling and determine its impact on osteolytic activity of breast cancer cells. Specific Aim 3: Determine the pre-clinical therapeutic benefit of overcoming the impact of Hh signaling and the hypoxic bone microenvironment to prevent osteolytic metastases of breast cancer. Expected outcome: The hypoxic bone microenvironment may select for tumor cells with aberrantly activated Hh signaling that are addicted to the hypoxic bone milieu for their sustenance. We will score the therapeutic benefit of combining a pharmacologic Hh inhibitor from Bristol-Myers Squibb in conjunction with a HIF-1 inhibitor, in a pre-clinical model. Impact: We anticipate that our work will motivate and guide the design of a Phase I/II clinical trial for breast cancer to determine th clinical effectiveness of a combined treatment regimen using Hh and hypoxia inhibitors in preventing and/or treating breast cancer osteolytic metastasis.